We have continued studying T cell apoptosis using a series of cell-permeable fluorogenic peptide substrates. Previous studies of thymocytes showed that Fas crosslinking triggered a caspase cascade in which the IETD (caspase 8 selective) substrate was the earliest activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when we examined apoptosis induced by anti-Fas in cytotoxic T lymphocytes by this approach, IETDase activation unexpectedly followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9,1, and 6). When examined by confocal microscopy, anti-Fas treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of sub-membrane proteins, leading to generation of membrane vesicles which are highly enriched in active caspase 8.